The principal aim of this HIVRAD Proposal is to identify one or more novel HIV-1 envelope (Env) antigens, identified by use of directed molecular evolution, that are capable of inducing broadly neutralizing antibody responses to primary isolates of the HIV-1 virus when tested in rabbits. Such antigens will ultimately be candidates for extensive testing in non-human primates and humans. A vaccine that induces neutralizing antibodies to the virus could potentially block infection or slow the development of AIDS. Starting with a single subtype B viral Env sequence, more immunogenic glycosylation variants will be identified. Subsequently, in vitro multigene recombination and in vivo screening will be performed. We will determine the ability of the Envelope variants to induce neutralizing antibodies that cross-react with various primary isolates of HIV-1. Immunization will be carried out using electroporation-facilitated DNA priming followed by protein boosting. A high-throughput viral neutralization assay will be employed to rapidly evaluate the results of animal immunization on panels of primary viral isolates. By testing numerous immunogens in rabbits during the course of this work, we will rigorously determine whether it is possible to create a pan-reactive immunogen, capable of inducing an immune response that can broadly neutralize HIV-1. The conclusion could greatly influence the direction of future HIV-1 vaccine research and development. A second aim of this work is to provide new and detailed structural information on the interaction of the HIV-1 gp120 Env protein with monoclonal antibodies that have the ability to broadly neutralize primary viral isolates. This will be accomplished by determination of three-dimensional structures of antibody-Env complexes, using rapid methods for high-throughput crystallization. In a third aim, various formats of Env will be tested to determine which one is optimal for presenting the most immunogenic Env sequences. Virus-like particles in which the Env complex is stabilized will be targeted.